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EP3676295B1 - Profilage phénotypique de cellules tumorales circulantes d'un carcinome hépatocellulaire pour la sélection d'un traitement - Google Patents

Profilage phénotypique de cellules tumorales circulantes d'un carcinome hépatocellulaire pour la sélection d'un traitement Download PDF

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EP3676295B1
EP3676295B1 EP18851615.7A EP18851615A EP3676295B1 EP 3676295 B1 EP3676295 B1 EP 3676295B1 EP 18851615 A EP18851615 A EP 18851615A EP 3676295 B1 EP3676295 B1 EP 3676295B1
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ctcs
hcc
vimentin
ctc
patients
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EP3676295A1 (fr
EP3676295A4 (fr
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Vatche AGOPIAN
Hsian-Rong Tseng
Shuang HOU
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University of California
University of California Berkeley
University of California San Diego UCSD
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University of California San Diego UCSD
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the invention provides a method of detecting hepatocellular carcinoma recurrence or metastasis, the method comprising:
  • the invention further provides a method of measuring markers of cirrhosis, hepatocellular carcinoma recurrence or metastasis in a blood sample obtained from a subject, the method comprising:
  • the invention also provides a method of identifying patients eligible for liver transplant, the method comprising performing the method above.
  • the invention further provides a method of screening for advanced or metastatic hepatocellular carcinoma in a subject, the method comprising performing either of the above methods.
  • the assay reagents, kits, and methods described herein overcome problems relating to poor discrimination of markers for tumor biology and in current clinicopathologic staging systems, as well as addressing other needs relating to the care and treatment of patients suffering from liver cancer and/or cirrhosis.
  • the disclosure herein provides methods of detecting hepatocellular carcinoma recurrence or metastasis, and of measuring markers of cirrhosis, hepatocellular carcinoma, including markers of hepatocellular carcinoma recurrence or metastasis, in a blood sample obtained from a subject.
  • the methods described herein accurately discriminate early-stage, liver transplant eligible patients from locally advanced, metastatic, liver transplant ineligible patients.
  • bound or “binding” refer to direct binding, e.g., antibody to antigen, as well as to indirect binding, such as may occur when a biotinylated antibody is bound to a streptavidin-coated surface via biotin-streptavidin binding.
  • a “microarray” is a linear or two-dimensional array of preferably discrete regions, each having a defined area, formed on the surface of a solid support.
  • label refers to a composition capable of producing a detectable signal indicative of the presence of the target in an assay sample. Suitable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • support refers to conventional supports such as beads, particles, dipsticks, fibers, filters, membranes and silane or silicate supports such as glass slides.
  • a "biological sample” refers to a sample of tissue or fluid isolated from an individual, including but not limited to, for example, blood, plasma, serum, tumor biopsy, urine, stool, sputum, spinal fluid, pleural fluid, nipple aspirates, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, cells (including but not limited to blood cells), tumors, organs, and also samples of in vitro cell culture constituent.
  • the sample is blood or other sample containing circulating cells, such as a fraction that includes peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • antibody is meant to be an immunoglobulin protein that is capable of specifically binding an antigen.
  • Antibody as used herein is meant to include antibody fragments, e.g. F(ab')2, Fab', Fab, capable of binding the antigen or antigenic fragment of interest.
  • to "prevent” or “protect against” a condition or disease means to hinder, reduce or delay the onset or progression of the condition or disease.
  • to "treat" a condition or disease means to ameliorate symptoms of the condition or disease.
  • Circulating tumor cells are isolated and selected on the basis of vimentin positive status as an indicator of disease status and severity.
  • the method comprises: (a) isolating circulating tumor cells (CTCs) by contacting a blood sample obtained from the subject with a set of capture antibodies, wherein the capture antibodies specifically bind asialoglycoprotein receptor (ASGPR), Glypican-3, and epithelial cell adhesion molecule (EpCAM).
  • the method further comprises: (b) contacting the isolated CTCs with an antibody that specifically binds vimentin.
  • the method further comprises: (c) measuring the number of vimentin-positive CTC per 4 ml blood of the sample, wherein hepatocellular carcinoma recurrence or metastasis is detected if 2 or more vimentin-positive CTCs per 4 ml blood are present in the sample.
  • the method further comprises: (c) assigning a status score that reflects the measured amount of vimentin-positive isolated CTCs per volume of blood sample.
  • the vimentin-positive cells are cytokeratin-positive and CD45-negative.
  • the method further comprises (d) referring the subject for surgical transplant.
  • the subject is referred for transplant if the status score is less than 2 vimentin-positive CTC per 4 ml blood.
  • the subject is referred for treatment for hepatocellular carcinoma if the status score is greater than or equal to 2 vimentin-positive CTCs per 4 ml blood.
  • the methods described herein can optionally further comprise referring the subject for treatment for advanced or metastatic hepatocellular carcinoma.
  • the invention additionally provides a method of screening for advanced or metastatic hepatocellular carcinoma in a subject.
  • the method comprises performing the method steps recited above.
  • a method of identifying patients eligible for liver transplant comprises performing the method steps recited above.
  • the invention further provides a method of measuring markers of cirrhosis or other liver disease in a subject.
  • the method comprises: (a) isolating circulating tumor cells (CTCs) by contacting a blood sample obtained from the subject with a set of capture antibodies, wherein the capture antibodies specifically bind asialoglycoprotein receptor (ASGPR), Glypican-3, and epithelial cell adhesion molecule (EpCAM); (b) contacting the isolated CTCs with an antibody that specifically binds vimentin; (c) measuring the amount of vimentin-positive isolated CTCs per volume of blood sample; and (d) treating the subject with liver transplant if the status score is less than 2 vimentin-positive CTC per 4 ml blood, or treating the subject for hepatocellular carcinoma if the status score is greater than or equal to 2 vimentin-positive CTCs per 4 ml blood.
  • CTCs circulating tumor cells
  • steps (a) and (b) can be performed separately or concomitantly.
  • the contacting of steps (a) and/or (b) are performed using a surface, such as a microarray or nanosurface, to which the antibodies are bound.
  • the nanosurface can be prepared as a microfluidic, antibody based CTC enrichment assay. This assay serves to efficiently capture and characterize CTC phenotypes of prognostic importance in HCC.
  • the modular NanoVelcro CTC platform has engrafted streptavidin on its nanosurface, allowing for any biotinylated cell-surface antibody to be employed for the capture of CTCs.
  • Those skilled in the art will appreciate other means by which antibodies can be used to capture CTCs, including other means of binding to a solid support and other means of detection.
  • the contacting of step (a) is performed with all of the capture antibodies bound to a single surface.
  • the capture antibodies are bound to the surface via biotin-streptavidin binding, such as by use of biotinylated antibodies that bind to a streptavidin-coated surface.
  • the contacting of step (a) is performed separately with each of the capture antibodies.
  • a microarray is used to facilitate detection of specific binding.
  • specific binding is detected through the use of distinct markers or labels.
  • immunoassay techniques such as enzyme immunoassays, microarray assays, and nanosurface assays.
  • Other assays can be employed, as will be understood to those skilled in the art.
  • representative examples of the sample include, but are not limited to, blood, plasma or serum, and other bodily fluids.
  • This Example demonstrates a novel "liquid-biopsy” assay capable of detecting HCC circulating tumor cells (CTCs), and characterizing phenotypic subpopulations with prognostic significance.
  • CTCs HCC circulating tumor cells
  • an antibody cocktail targeting the cell-surface markers asialoglycoprotein receptor (ASGPR), Glypican-3, and epithelial cell adhesion molecule (EpCAM) was optimized for HCC-CTC capture utilizing the NanoVelcro microfluidic assay.
  • HCC-CTCs and vimentin(+)-CTCs (a subpopulation expressing an epithelial-to-mesenchymal phenotype) to accurately discriminate tumor stage, recurrence, progression, and overall survival was evaluated in a prospective study of 80 patients.
  • Hepatocellular carcinoma is the fifth most common cancer and the second most common cause of cancer related death worldwide.
  • current clinicopathologic staging systems and serum biomarkers e.g. alpha-fetoprotein, AFP
  • AFP alpha-fetoprotein
  • LT liver transplantation
  • predictors of response remain unavailable.
  • Circulating tumor cells are thought to originate from the primary tumor or metastatic sites, can be detected in the peripheral blood, and are implicated as a potential cause of post-surgical recurrence and metastases.
  • CTCs Circulating tumor cells
  • studies evaluating CTCs in HCC have found limited utility.
  • CTC enrichment assays including the FDA-approved CellSearch TM CTC assay, rely on the use of antibodies against the epithelial cell-surface marker EpCAM to "capture" CTCs by antigen-specific immunomagnetic separation from leukocytes.
  • NanoVelcro CTC assay combines a microfluidic system with enhanced topographic interactions and CTC-capture antibody coated nanostructured substrates to allow for the efficient separation and capture of HCC-CTCs from background WBCs.
  • NanoVelcro CTC Assay The working principle of the NanoVelcro CTC Assay has been utilized for many solid tumors, including prostate cancer, melanoma, and pancreatic cancer.(17,18)
  • HCC CTC capture and immunostaining antibodies(7,19) were investigated, followed by validation of their efficacy using a HCC tissue microarray (TMA), HCC cell lines, and a pilot group of HCC patients.
  • TMA HCC tissue microarray
  • CTCs relies on the antigen-specific immobilization of CTCs on the NanoVelcro surface using antibodies directed against HCC cell surface markers. Immobilized CTCs are then identified and phenotyped based on their immunohistochemical staining characteristics ( Fig. 7 ). A wide selection of antibodies for both CTC capture, as well as CTC identification and phenotyping, were evaluated using an 8 HCC cell line panel as detailed in the supplemental methods.
  • TMA tissue microarray
  • Staining intensity was assessed on a 4-point scale (none/weak/moderate/strong staining) by a single pathologist (S.W.F.), and tumors were considered to have stained positive if they displayed moderate or strong staining to any one of the antibodies tested ( Fig. 9 ). TMA staining results were summarized as shown in Fig.1A .
  • the NanoVelcro CTC assay utilizes a microfluidic chaotic mixer to enhance CTC interactions with the capture antibody coated nanosubstrate surface of the chip to enhance CTC capture rates ( Fig. 7A ). Following CTC capture, CTCs are immunostained as outlined in our workflow ( Fig. 7B ) and then identified and phenotyped using multi-color ICC and cytometric assessment ( Figs. 10 & 11 ). While the performance of the NanoVelcro CTC assay has been reported for many solid tumors, we first validated the methodology for HCC using cell lines as previously described and detailed in the supplemental methods.(21) All experiments were performed at the optimum flow rate of 1 mL per hour. ( Fig. 12A ).
  • HCC non-malignant liver disease
  • IRB #14-001932 patients with HCC were enrolled (IRB #14-001932).
  • Inclusion criteria included pathologic or radiographic (LIRADS-5) diagnosis of HCC, with patients having synchronous or past (within 5 years) extrahepatic malignancies excluded from enrollment.
  • a database of demographic and clinicopathologic information was maintained prospectively, with clinical staging assigned based on either pathologic (when available) or radiographic assessment (Milan criteria [MC](22) and University of California, San Francisco [UCSF](23) transplant criteria) (Table 1) at study enrollment.
  • WBCs When analyzing the multichannel ICC image, WBCs were defined as round/ovoid cells, DAPI+/CD45+/CK-, with size ⁇ 6- ⁇ m; and HCC-CTCs are defined as round/ovoid cells, DAPI+/CD45-/CK+, with size ⁇ 6- ⁇ m.
  • Epithelial-to-mesenchymal phenotype, vimentin(+)-CTCs are the subpopulation of HCC-CTCs defined as round/ovoid events, DAPI+/CD45-/CK+/vimentin+, with size ⁇ 6- ⁇ m. Any CD45 positivity greater than 2x background discounted a cell as being a CTC.
  • CTCs were enumerated by the same blinded researcher (S.H.) and CTC counts are represented as a total count per 4-mL VB ( Fig.2B ).
  • Time to recurrence was evaluated only in the subset of early stage, potentially curable patients undergoing curative intent surgical or locoregional therapy, where the initial post-treatment imaging showed no viable arterially enhancing lesions (irrespective of surgical resection, ablation, or in select cases, TACE treatment).
  • Recurrence was defined as a new or recurrent radiographic lesion meeting OPTN criteria for HCC.
  • Time to recurrence (TTR) of radiographically evident disease, progression-free (PFS), and overall survival (OS) were computed using Kaplan-Meier methods and compared by the log-rank (Mantel-Cox) test.
  • Hepatocellular carcinoma cell lines (HepG2, Hep3B, Huh-7, PLC/PRF/5, SNU-387, HLE, SNU-449, SK-Hep-1, and MCF-7) were obtained from American Type Culture Collection (ATCC, Manassas, VA), grown using ATCC recommended media at 37C with 5% CO 2 , and routinely passaged at 80% confluence using an iso-osmotic sodium citrate solution for cell release (Thermo Fisher Scientific, Waltham, MA). The cell lines were specifically selected to ensure a broadly representative class of HCC cells with varying phenotypes, differentiation, sites of origin, and cell surface marker expressions (Table 2). Table 2.
  • CTC capture antibodies For the initial calibration experiments, 500 cells from each of the cell lines were first incubated with CTC capture antibodies. After briefly washing, the pre-labeled HCC cell line cells were spiked into 2-mL healthy donor blood to create artificial blood samples. Artificial blood samples were run in triplicate at flow rates of 0.1, 0.5, 1.0, 2.0, and 3.0 mL/h using 8 of the HCC cell lines (Hep-G2, PLC/PRF5, Huh-7, Hep-3B, SNU387, HLE, SNU449, and SK-Hep1). CTC capture efficiency, defined as the fraction of CK+/CD45- CTCs captured and identified over the known total number of spiked cells, was maximized at a flow rate of 1.0 ml/h ( Figure 12 ). This optimized flow rate was used for all subsequent experiments. The capture antibodies used, either individually or in combination, did not substantially affect the optimum flow rate of 1 mL per hour.
  • the triple antibody cocktail of EpCAM, ASGPR, and GPC-3 was assessed for CTC capture efficiency.
  • each of the 8 HCC cell lines were run on 4 NanoVelcro chips: one chip for each individual antibody with the fourth assay evaluating the combination of all 3 antibodies simultaneously. The cell capture efficiencies were compared to determine the optimum capture antibody cocktail ( Fig. 1B & C ).
  • VB venous blood
  • ACD solution A tubes BD-Pharmigen, Franklin Lakes, NJ
  • All samples were processed within 24 hours of collection.
  • Mononuclear cells were isolated from the blood samples by density gradient centrifugation. Blood samples were first diluted 1:1 with PBS (Sigma, St. Louis, MO) and transferred to 12 mL LeucoSep centrifuge tubes (VWR, Radnor, PA) with 3 mL of Histopaque-1077 (Sigma) below the porous barrier. Samples were centrifuged at 400 ⁇ g at 4C for 30 minutes with the break off.
  • the buffy coat layer was then transferred to a new 15 mL tube and washed with 5 mL of wash medium (RPMI with 5% FBS, Gibco, Carlsbad, CA), centrifuged at 300 x g for 10 minutes at 4C, and the pellet resuspended in 200 ⁇ L of PBS supplemented with 2% donkey serum (DS). 6 ⁇ L of biotinylated capture antibody cocktail (EpCAM, ASGPR, GPC-3) was added and the mixture was incubated on a shaker for 30 minutes at room temperature. Following a final wash step, the pellet was re-suspended in 200 ⁇ L of PBS and immediately processed on the NanoVelcro platform.
  • wash medium RPMI with 5% FBS, Gibco, Carlsbad, CA
  • DS donkey serum
  • NanoVelcro chips were assembled and operated as previously described.(4) Prepared samples were injected into the device at the optimized flow rate of 1.0 mL/h, followed by 4% paraformaldehyde (PFA) injected at the same rate for fixation. The chips were then removed from the NanoVelcro platform and washed in PBS for 15 minutes. Chips were blocked and permeabilized using PBS + 2% DS + 0.1% Triton X-100 (Sigma) for 15 minutes. Chips were then incubated with a cocktail of primary antibodies including two mouse anti-CD45 antibodies (DAKO, Abcam), two rabbit anti-cytokeratin (CK) antibodies (DAKO, Abcam) and 1 chicken anti-vimentin antibody (Abcam).
  • DAKO mouse anti-CD45 antibodies
  • CK rabbit anti-cytokeratin
  • Abcam 1 chicken anti-vimentin antibody
  • Chips were first scanned at 40x power by an automated chip scanning protocol using the NIS Elements 4.1 software (Nikon, Tokyo, Japan) on an Eclipse 90i fluorescent microscope to identify candidate cells. Higher magnification manual imaging of candidate cells was then performed at 400x power to verify results.
  • WBCs were defined as round/ovoid, DAPI+, CD45+ and CK-.
  • CTCs were defined as round/ovoid, size ⁇ 6- ⁇ m, DAPI+, CD45-, and CK+ and/or vimentin+. Any CD45 positivity greater than 2x background discounted a cell as being a CTC.
  • Final CTC counts are represented as a total count per 4 mL VB.
  • CTCs are first incubated with biotinylated antibody cocktail (EpCAM, ASGPR, GPC-3) and then "captured" by the NanoVelcro assay through the interaction of biotin on the CTCs with the streptavidin-coated surface of the NanoVelcro chips.
  • EpCAM biotinylated antibody cocktail
  • GPC-3 biotinylated antibody cocktail
  • the CTC capture efficiency for each individual capture antibody was compared to the combination of all 3 antibodies for 8 HCC cell lines ( Fig.1B ).
  • HCC-CTCs were stained with CK as the primary epithelial marker, and co-stained with hepatocyte-specific markers AFP, arginase, and hep-par-1. AFP, arginase, and hep-par-1 staining was only noted in the CK+ cells and never in the CD45+ leukocytes, confirming the specificity of our ICC criteria for HCC-CTCs ( Fig. 11 ). Additionally, we discovered a subpopulation of vimentin(+) CTCs with an epithelial-to-mesenchymal phenotype. Based on these initial experiments, all prospective HCC patients enrolled in the study underwent enumeration of HCC-CTCs and the subpopulation of vimentin(+)-CTCs ( Fig.2 ).
  • PFS blood pressure
  • Vimentin(+) CTCs were found in 31(50.8%) patients with HCC (median:1, range:0-20) and never in patients with NMLD or healthy controls ( Fig.4C ).
  • the number of vimentin(+)-CTCs correlated with increasing tumor stage, with only 4/31(12.9%) early stage patients demonstrating vimentin(+)-CTCs (median:0, IQR:0-0, range:0-12), compared to 18/21(85.7%) locally advanced patients (median:4, IQR:2-6, range:0-8), and 9/9(100%) metastatic HCC patients (median:8, IQR:8-14, range:1-20).
  • Significantly more vimentin(+)-CTCs were found in patient with radiographic evidence of portal vein invasion (p ⁇ 0.001).
  • HCC-CTCs and vimentin(+)-CTCs as biomarkers goes beyond initial prognosis, presenting potential utility for both longitudinal disease monitoring as well as appropriate treatment selection.
  • 11 patients in the study underwent serial blood draws and CTC enumeration over the course of treatment, with disappearance of CTCs following successful tumor resection and ablation, and a subsequent reappearance of CTCs prior to clinical recurrence (data not shown).
  • One such example is illustrated in Fig.6A .
  • the patient was a 63-year-old woman with compensated cirrhosis and a 5.8 cm arterially enhancing biopsy-proven HCC.
  • vimentin(+)-CTCs consistently portended faster time to recurrence following locoregional treatment (Fig.5D), with an example illustrated in Fig.6B .
  • the patient had a solitary 4cm right hepatic lobe HCC without evidence of metastases, but despite being radiographically staged as an early stage patient, was found to have 12 vimentin(+) CTCs (15 HCC-CTCs).
  • HCC-CTCs vimentin(+) CTCs
  • CTCs are emerging as a promising biomarker for several cancer types, but their application to HCC has been limited when utilizing existing assays that rely on epithelial cell-surface markers alone for CTC capture.
  • we present the development of a novel multimarker CTC capture platform that allows for the identification, enumeration, and analysis of HCC-CTCs with high sensitivity and specificity.
  • we identify a phenotypic subpopulation of vimentin(+)-CTCs which are highly associated with advanced or metastatic HCC, increased recurrence after potentially curative therapy, and inferior progression-free and overall survival.
  • the majority of current CTC enrichment platforms rely on antibodies to the cell-surface marker EpCAM alone, potentially limiting their utility in HCC where expression of EpCAM is reported in only 20-35% of tumors.
  • ASGPR a transmembrane cell-surface protein highly expressed in well-differentiated HCCs, yielded high CTC capture rates in previous HCC studies, and was used in our antibody cocktail.(7,9,19)
  • GPC-3 Based on our TMA studies, we also included GPC-3, since its expression has been associated with the presence of poorly-differentiated HCC,27 arguably the most important subset of tumors to be detected given their poor prognosis.
  • This unique three-marker antibody cocktail allowed us to detect CTCs from 96.7% of all patients with HCC, compared to 20-50% captured using EpCAM alone.(6,28-30)
  • Vimentin is an intermediate filament ubiquitously expressed in normal mesenchymal cells and is involved in cellular processes including stress resistance and structural integrity.
  • Vimentin overexpression is widely regarded as the canonical marker of EMT in epithelial cancers (31), and tumors expressing vimentin demonstrate accelerated tumor growth and increased invasiveness.
  • vimentin overexpression in the primary tumor has been linked to more aggressive tumor biology, inferior survival outcomes, and the establishment of the tumor initiating capacity critical for metastases.
  • vimentin overexpression in CTCs has been established previously in several cancer types, but the utility of vimentin for HCC-CTCs has only recently been considered.(13,32-34)
  • vimentin(+)-CTCs were found almost exclusively in patients with advanced stage HCC corroborates previous reports in breast and prostate cancer that found the presence of vimentin(+)-CTCs as a marker of metastatic disease and worse outcomes.
  • cytokeratin-positive epithelial cells have been found in the blood of patients with inflammatory gastrointestinal disease such as inflammatory bowel disease and cirrhosis, the use of additional markers can ensure that no benign epithelial cells are being mis-identified as CTCs.(36) This is one reason that vimentin(+)-CTCs may hold more clinical relevance for HCC, where the vast majority of patients have underlying liver cirrhosis.
  • vimentin(+)-CTCs vastly outperform the only existing serum biomarker, AFP, for both HCC staging and prognosis ( Fig.4D ).
  • the finding that vimentin(+)-CTCs are highly predictive of earlier recurrence and worse survival in otherwise indistinguishable early stage patients undergoing locoregional or surgical therapy highlights their potential utility as a biomarker for liver transplant candidate selection.
  • the use of vimentin(+)-CTCs as an adjunct biomarker may help overcome the limitation of existing clinicopathologic staging systems in distinguishing cancer-specific outcomes within the subset of potentially curable patients.
  • HCC-CTCs as a tool for both longitudinal monitoring of disease and identification of patients with aggressive underlying disease is apparent. Given the ease and repeatability of the non-invasive HCC-CTC assay, the reliable serial monitoring of a patient's response to treatment would be possible. In patients undergoing surgical resection and ablation, total HCC-CTCs dropped significantly after treatment, but increased in the subset of patients who subsequently proved to have recurrence on follow up imaging ( Fig.6 ). In addition, Vimentin(+)-CTCs were highly predictive of recurrence and progression in the subset of potentially curable patients undergoing successful locoregional therapy who initially demonstrated no evidence of disease on post-treatment imaging. This highlights the potential role of vimentin(+)-CTCs for the selection of patients undergoing curative liver transplantation, where the identification of patients likely to have poor outcomes is a critical, unmet need to avoid the loss of scarce donor allografts.
  • the HCC multimarker antibody-based CTC capture assay described herein allows for the identification of HCC-CTCs as well as a distinct subpopulation of vimentin(+)-CTCs.
  • the study first optimized and validated the assay's functionality for capturing HCC-CTCs using both spiked cell line and clinical samples.
  • the assay allowed for detection of HCC-CTCs from nearly all patients with HCC, with highly accurate discrimination between patients with HCC and those with NMLD or healthy controls.

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Claims (10)

  1. Procédé de détection d'une récidive ou d'une métastase de carcinome hépatocellulaire, le procédé comprenant :
    (a) l'isolation de cellules tumorales circulantes (CTC) à partir d'un échantillon de sang obtenu d'un sujet en mettant en contact les CTC avec un ensemble d'anticorps de capture, dans lequel les anticorps de capture se lient spécifiquement au récepteur de l'asialoglycoprotéine (ASGPR), au Glypican-3 et à la molécule d'adhérence des cellules épithéliales (EpCAM) ;
    (b) la mise en contact des CTC isolées avec un anticorps qui se lie spécifiquement à la vimentine ;
    (c) la mesure du nombre de CTC positives pour la vimentine par 4 ml de sang de l'échantillon, dans lequel la rédicive ou la métastase de carcinome hépatocellulaire est détectée si 2 CTC positives pour la vimentine ou plus pour 4 ml de sang sont présentes dans l'échantillon.
  2. Procédé de mesure de marqueurs de cirrhose, de récidive ou de métastase de carcinome hépatocellulaire dans un échantillon de sang prélevé sur un sujet, le procédé comprenant :
    (a) l'isolation de cellules tumorales circulantes (CTC) en mettant en contact l'échantillon de sang avec un ensemble d'anticorps de capture, dans lequel les anticorps de capture se lient spécifiquement au récepteur de l'asialoglycoprotéine (ASGPR), au Glypican-3 et à la molécule d'adhérence des cellules épithéliales (EpCAM) ;
    (b) la mise en contact des CTC isolées avec un anticorps qui se lie spécifiquement à la vimentine ;
    (c) l'attribution d'un score d'état qui reflète la quantité mesurée de CTC isolées positives pour la vimentine par volume d'échantillon de sang ; et, éventuellement,
    (d) l'orientation du sujet vers une greffe chirurgicale si le score d'état est inférieur à 2 CTC positives pour la vimentine pour 4 ml de sang, ou l'orientation du sujet vers un traitement pour un carcinome hépatocellulaire si le score d'état est supérieur ou égal à 2 CTC posivies pour la vimentine pour 4 ml de sang.
  3. Procédé de dépistage d'un carcinome hépatocellulaire avancé ou métastatique chez un sujet, le procédé comprenant la mise en œuvre du procédé selon la revendication 1 ou 2.
  4. Procédé selon l'une quelconque des revendications 1 à 3, comprenant en outre l'orientation du sujet vers un traitement pour un carcinome hépatocellulaire avancé ou métastatique.
  5. Procédé d'identification de patients éligibles à une greffe du foie, le procédé comprenant la mise en œuvre du procédé selon la revendication 2.
  6. Procédé selon la revendication 1 ou 2, dans lequel la mise en contact des étapes (a) et/ou (b) est effectuée à l'aide d'un microréseau ou d'une nanosurface à laquelle les anticorps sont liés.
  7. Procédé selon la revendication 1, 2 ou 3, dans lequel la mise en contact de l'étape (a) est effectuée avec tous les anticorps de capture liés à une seule surface.
  8. Procédé selon la revendication 7, dans lequel les anticorps de capture sont liés à la surface via une liaison biotine-streptavidine.
  9. Procédé selon la revendication 1, 2 ou 3, dans lequel la mise en contact de l'étape (a) est effectuée séparément avec chacun des anticorps de capture.
  10. Procédé selon l'une quelconque des revendications 1 à 9, dans lequel les cellules positives pour la vimentine sont positives pour la cytokératine et négatives pour le CD45.
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